Stabilized pharmaceutical composition in lyophilized form

ABSTRACT

A stabilized pharmaceutical composition in lyophilized form comprising: a cyclic polypeptide compound represented by the general formula (I): 
                         
wherein R 1  is a hydrogen atom or an acyl group and R 2  and R 3  are, the same or different, a hydrogen atom or a hydroxyl group, or a salt thereof and the stabilizer.

This application is a divisional of U.S. Ser. No. 09/786,125, filed Mar.1, 2001, now U.S. Pat. No. 6,774,104, which is the national-stage under35 U.S.C. §371 of PCT/JP00/04381, filed Jun. 29, 2000. This applicationalso claims priority to JAPAN 11/187713, filed Jul. 1, 1999.

TECHNICAL FIELD

The present invention relates to a stabilized pharmaceutical compositionin lyophilized form containing a cyclic polypeptide compound. Moreparticularly, the present invention relates to a stabilizedpharmaceutical composition in lyophilized form containing a cyclicpolypeptide compound or its pharmaceutically acceptable salt and astabilizer.

The cyclic polypeptide compound of the present invention is representedby the general formula (I):

wherein R¹ is a hydrogen atom or an acyl group and R² and R³ are, thesame or different, a hydrogen atom or a hydroxyl group. The compound hasan antimicrobial activity, particularly an antifungal activity and aβ-1,3-glucan synthase inhibiting action, and is useful for preventingand treating various kinds of infectious diseases including Pneumocystiscarinii infection, e.g., carinii pneumonia.

BACKGROUND ART

Among the cyclic polypeptide compounds represented by the above formula(I), a compound wherein R¹ is a hydrogen atom and R² and R³ are hydroxylgroups and a compound wherein R¹, R² and R³ are hydrogen atoms areobtained by a fermentation process disclosed by European Patent No.0462531 and processes disclosed by WO97/32975 and by WO97/47738. Acompound wherein R¹ is an acyl group and its production process aredisclosed by U.S. Pat. Nos. 5,376,634 and 5,569,646 and WO96/11210 andWO99/40108.

The cyclic polypeptide compounds (I) and their salts are generallyunstable to light, humidity, acids, heat and the like. Therefore,desired is development of pharmaceutical preparations in which thecyclic polypeptide compounds and their salts are stabilized.

DISCLOSURE OF INVENTION

The present invention provides a stabilized pharmaceutical compositionin lyophilized form containing a cyclic polypeptide compound (I) or itspharmaceutically acceptable salt and a stabilizer.

The “acyl group” for R¹ in the formula (I) representing the cyclicpolypeptide compound of the present invention is now explained. In thecontext of the present specification, “lower” means having one to sixcarbon atoms unless otherwise indicated.

As examples of the acyl group, may be mentioned aliphatic acyl groups,aromatic acyl groups, aromatic-aliphatic acyl groups and heterocyclicacyl groups derived from aliphatic, aromatic, aromatic-aliphatic andheterocyclic carboxylic acids.

Examples of the aliphatic acyl groups include lower or higher alkanoylgroups such as formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl,pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl,nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl,icosanoyl, etc.; cycloalkanoyl groups such as cyclopentanoyl andcyclohexanoyl; lower alkoxycarbonyl groups such as methoxycarbonyl,ethoxycarbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl,heptyloxycarbonyl, etc.; lower alkanesulfonyl groups such asmethanesulfonyl, ethanesulfonyl, etc.; lower alkoxysulfonyl groups suchas methoxysulfonyl, ethoxysulfonyl, etc.; and the like.

Examples of the aromatic acyl groups include aroyl groups such asbenzoyl, toluoyl, naphthoyl and the like.

Examples of the aromatic-aliphatic acyl groups include ar(lower)alkanoylgroups such as phenyl(C₁–C₆)alkanoyl (e.g., phenylacetyl,phenylpropanoyl, phenylbutanoyl, phenylisobutanoyl, phenylpentanoyl,phenylhexanoyl, etc.), naphthyl(C₁–C₆)alkanoyl (e.g., naphthylacetyl,naphthylpropanoyl, naphthylbutanoyl, etc.) and the like;ar(lower)alkenoyl group such as phenyl(C₃–C₆)alkenoyl (e.g.,phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl,phenylhexenoyl, etc.), naphthyl(C₃–C₆)alkenoyl (e.g., naphthylpropenoyl,naphthylbutenoyl, etc. and the like;

-   ar (lower)alkoxycarbonyl groups such as phenyl(C₁–C₆)alkoxycarbonyl    (e.g., benzyloxycarbonyl, etc.), fluorenyl(C₁–C₆)alkoxycarbonyl    (e.g., fluorenylmethoxycarbonyl, etc.) and the like;-   aryloxycarbonyl groups such as phenoxycarbonyl, naphthoxycarbonyl,    etc.;-   aryloxy(lower)alkanoyl groups such as phenoxyacetyl,    phenoxypropionyl, etc.;-   arylcarbamoyl groups such as phenylcarbamoyl, etc.;-   arylthiocarbamoyl groups such as phenylthiocarbamoyl, etc.;-   arylglyoxyloyl groups such as phenylglyoxyloyl, naphthylglyoxyloyl,    etc.;-   arylsulfonyl groups which may be optionally substituted by a lower    alkyl group such as phenylsulfonyl, p-tolylsulfonyl, etc.; and the    like.

Examples of the heterocyclic acyl groups include heterocyclic carbonylgroups such as thenoyl, furoyl, nicotinoyl, etc.;

-   heterocyclic(lower)alkanoyl groups such as heterocyclic acetyl,    heterocyclic propanoyl, heterocyclic butanoyl, heterocyclic    pentanoyl, heterocyclic hexanoyl, etc.;-   heterocyclic(lower)alkenoyl groups such as heterocyclic propenoyl,    heterocyclic butenoyl, heterocyclic pentenoyl, heterocyclic    hexenoyl, etc.;-   heterocyclic glyoxyloyl and the like.

The acyl group for R¹ may have one or more suitable substituent(s).Among the above-mentioned examples for the acyl groups, an aroyl groupwhich may have one or more suitable substituent(s) is particularlypreferable.

Examples of suitable substituents in the acyl group include aheterocyclic group substituted by an aryl group having a lower alkoxygroup, a heterocyclic group substituted by an aryl group having a loweralkoxy(lower)alkoxy group, a heterocyclic group substituted by an arylgroup having a lower alkoxy(higher)alkoxy group, a heterocyclic groupsubstituted by an aryl group having a cyclo(lower)alkyloxy group, aheterocyclic group substituted by an aryl group having a heterocyclicgroup, a heterocyclic group substituted by a cyclo(lower)alkyl grouphaving a cyclo(lower)alkyl group, a heterocyclic group substituted by anaryl group having an aryl group substituted by a loweralkoxy(lower)alkoxy and a heterocyclic group substituted by an arylgroup having a heterocyclic group substituted by a cyclo(lower)alkylgroup.

Among these examples, preferred are an unsaturated 3- to 8-memberedheteromonocyclic group containing one to two oxygen atom(s) and one tothree nitrogen atom(s) and substituted by phenyl having (C₄–C₆)alkoxy,an unsaturated condensed heterocyclic group containing one to two sulfuratom(s) and one to three nitrogen atom(s) and substituted by phenylhaving (C₄–C₆)alkoxy, an unsaturated 3- to 8-membered heteromonocyclicgroup containing one to two sulfur atom(s) and one to three nitrogenatom(s) and substituted by phenyl having (C₁–C₄)alkoxy(C₄–C₆)alkoxy, anunsaturated 3- to 8-membered heteromonocyclic group containing one totwo sulfur atom(s) and one to three nitrogen atom(s) and substituted byphenyl having (C₁–C₄)alkoxy(C₇–C₁₄)alkoxy, a saturated 3- to 8-memberedheteromonocyclic group containing one to four nitrogen atom(s) andsubstituted by phenyl having (C₁–C₄)alkoxy(C₇–C₁₄)alkoxy, an unsaturatedcondensed heterocyclic group containing one to two sulfur atom(s) andone to three nitrogen atom(s) and substituted by phenyl havingcyclo(C₄–C₆)alkyloxy, an unsaturated condensed heterocyclic groupcontaining one to two sulfur atom(s) and one to three nitrogen atom(s)and substituted by phenyl, a saturated 3- to 8-membered heteromonocyclicgroup containing one to two oxygen atom(s) and one to three nitrogenatom(s), a saturated 3- to 8-membered heteromonocyclic group having oneto four nitrogen atom(s) and substituted by cyclo(C₄–C₆)alkyl havingcyclo(C₄–C₆)alkyl, an unsaturated 3- to 8-membered heteromonocyclicgroup having one to two sulfur atom(s) and one to three nitrogen atom(s)and substituted by phenyl having phenyl substituted by(C₁–C₄)alkoxy(C₁–C₄)alkoxy, an unsaturated 3- to 8-memberedheteromonocyclic group containing one to two sulfur atom(s) and one tothree nitrogen atom(s) and substituted by phenyl having a saturated 3-to 8-membered heteromonocyclic group which contains one to four nitrogenatom(s) and is substituted by cyclo(C₄–C₆)alkyl, and an unsaturatedcondensed heterocyclic group containing one to two sulfur atom(s) andone to three nitrogen atom(s) and substituted by phenyl having asaturated 3- to 8-membered heteromonocyclic group which contains one tofour nitrogen atom(s) and has cyclo(C₄–C₆)alkyl.

Among these, particularly preferred are an isoxazolyl group substitutedby phenyl having pentyloxy, an imidazothiadiazolyl group substituted byphenyl having pentyloxy, a thiadiazolyl group substituted by phenylhaving methoxyhexyloxy, a thiadiazolyl group substituted by phenylhaving methoxyoctyloxy, a thiadiazolyl group substituted by phenylhaving methoxyheptyloxy, an imidazothiadiazolyl group substituted byphenyl having cyclohexyloxy, an imidazothiadiazolyl group substituted byphenyl having dimethylmorpholino, a piperazinyl group substituted byphenyl having methoxyheptyloxy, a piperazinyl group substituted byphenyl having methoxyoctyloxy, a piperazinyl group substituted bycyclohexyl having cyclohexyl, a thiadiazolyl group substituted by phenylhaving phenyl substituted by methoxyethoxy, a thiadiazolyl groupsubstituted by phenyl having phenyl substituted by methoxybutoxy, athiadiazolyl group substituted by phenyl having phenyl substituted byethoxypropoxy, an imidazothiadiazolyl group substituted by phenyl havingpiperazinyl substituted by cyclohexyl, an imidazothiadiazolyl groupsubstituted by phenyl having piperazinyl substituted by cyclohexyl, andthe like.

Accordingly, particularly suitable examples of the acyl group of R¹ maybe a benzoyl group having isoxazolyl substituted by phenyl havingpentyloxy, a benzoyl group having imidazothiadiazolyl substituted byphenyl having pentyloxy, a benzoyl group having thiadiazolyl substitutedby phenyl having methoxyhexyloxy, a benzoyl group having thiadiazolylsubstituted by phenyl having methoxyoctyloxy, a benzoyl group havingthiadiazolyl substituted by phenyl having methoxyheptyloxy, a benzoylgroup having imidazothiadiazolyl substituted by phenyl havingcyclohexyloxy, a benzoyl group having imidazothiadiazolyl substituted byphenyl having dimethylmorpholino, a benzoyl group having piperazinylsubstituted by phenyl having methoxyheptyloxy, a benzoyl group havingpiperazinyl substituted by phenyl having methoxyoctyloxy, a benzoylgroup having piperazinyl substituted by cyclohexyl having cyclohexyl, abenzoyl group having thiadiazolyl substituted by phenyl having phenylsubstituted by methoxyethoxy, a benzoyl group having thiadiazolylsubstituted by phenyl having phenyl substituted by methoxybutoxy, abenzoyl group having thiadiazolyl substituted by phenyl having phenylsubstituted by ethoxypropoxy, a benzoyl group having imidazothiadiazolylsubstituted by phenyl having piperazinyl substituted by cyclohexyl, abenzoyl group having imidazothiadiazolyl substituted by phenyl havingpiperazinyl substituted by cyclohexyl, and the like.

Particularly preferable examples of the acyl groups of R₁ arerepresented by the formulas:

The cyclic polypeptide compounds (I) having the above-mentioned acylgroups may be prepared from a compound having a hydrogen atom as R₁ andhydroxyl groups as R² and R³ or a compound having hydrogen atoms as R¹,R² and R³ according to the U.S. Pat. Nos. 5,376,634 and 5,569,646 andWO96/11210 and WO99/40108.

Suitable salts of the cyclic polypeptide compounds (I) are soluble inwater and pharmaceutically acceptable salts including salts with basesand acid addition salts. Such a salt may be prepared by treating thecyclic polypeptide compound (I) with an appropriate base or acidaccording to the conventional method.

As salts with bases, may be mentioned salts with inorganic bases such asalkali metal salts (e.g., sodium salts, potassium salts, etc.), alkalineearth metal salts (e.g., calcium salts, magnesium salts, etc.), ammoniumsalts and the like; salts with organic bases such as organic amine salts(e.g., triethylamine salts, diisopropylethylamine salts, pyridine salts,picoline salts, ethanolamine salts, triethanolamine salts,dicyclohexylamine salts, N,N′-dibenzylethylenediamine salts, etc.); andthe like.

As acid addition salts, may be mentioned inorganic acid addition salts(e.g., hydrochlorides, hydrobromides, sulfates, phosphates, etc.); andorganic carboxylic or sulfonic acid addition salts (e.g., formates,acetates, trifluoroacetates, maleates, tartrates, fumarates,methnesulfonates, benzenesulfonates, toluenesulfonates, etc.). Further,may also be mentioned salts with basic or acidic amino acids (e.g.,salts with arginine, aspartic acid, glutamic acid, etc.).

The cyclic polypeptide compounds (I) of the present invention alsoinclude possible conformers and a pair or more of stereoisomers such asgeometric isomers and optical isomers which may exist due to asymmetriccarbon atoms.

The preferable ones of the cyclic polypeptide compounds (I) arerepresented by the following formulas (II) to (VI):

(to be continued on the next page)

The most preferable one is represented by the formula (II).

The amount of the cyclic polypeptide compound (I) or itspharmaceutically acceptable salt contained in the composition for asingle unit dosage of the present invention is 0.1 to 400 mg, morepreferably 1 to 200 mg, still more preferably 10 to 100 mg, specifically10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95 and100 mg.

As the stabilizer, may be mentioned polysaccharides, disaccharides,sodium chloride and a combination thereof.

Examples of the polysaccharide are dextran, starch, cellulose andhyaluronic acid; and examples of the disacharide are lactose, maltoseand sucrose. The polysaccharide or disaccharide contained in thepharmaceutical composition of the present invention may beα-monohydrate, α-anhydride, β-anhydride or a combination thereof.

The amount of the stabilizer used in the pharmaceutical composition ofthe present invention should be at least sufficient for stabilizing thecyclic polypeptide compound (I) or its pharmaceutically acceptable saltin the composition. In order to stabilize the cyclic polypeptidecompound (I), one part by weight of the stabilizer with respect to onepart by weight of the cyclic polypeptide compound (I) or itspharmaceutically acceptable salt in the present composition issufficient at least. The stabilizer may also serve as a carrier or anexcipient. Thus the use amount of stabilizer does not have a particularupper limit and may be determined in consideration of the weight orvolume of the composition with respect to a unit dose of the compoundand the like. However, such amount is preferably 0.4 to 50 parts byweight, more preferably 0.6 to 20 parts by weight, still more preferably0.8 to 10 parts by weight with respect to one part by weight of thecyclic polypeptide compound (I) or its pharmaceutically acceptable salt,though it varies depending upon the kind and the used amount of thecyclic polypeptide compound (I) or its pharmaceutically acceptable salt,its preparation form and/or the like. Specifically, it is morepreferable that 1 to 20 parts, still more preferably 2 to 10 parts byweight of the disaccharide are used with respect to one part by weightof the cyclic polypeptide compound (I) or its pharmaceuticallyacceptable salt. Specifically, it is more preferable that 0.6 to 20parts, still more preferably 0.8 to 10 parts by weight of sodiumchloride are used with respect to one part by weight of the cyclicpolypeptide compound (I) or its pharmaceutically acceptable salt.

The pharmaceutical composition of the present invention may be producedaccording to methods known in the art with using additives if necessary.Here, Basic Lecture on Development of Pharmaceuticals XI 20 Productionof Pharmaceuticals (the second volume) (edited by Kyosuke Tsuda andHisashi Nogami and published by Chizyo Shoten) is mentioned forreference. The lyophilized composition may be obtained by preparing anaqueous solution of the cyclic polypeptide compound (I) or itspharmaceutically acceptable salt and the stabilizer, optionally adding apH adjustor (citric acid anhydrous, sodium hydroxide, etc.) as requiredto attain pH 4.0–7.5, preferably pH 4.5–7.0, and then lyophilizing theresulting solution in vial according to a conventional method. Thus, thestabilized pharmaceutical composition in lyophilized form, whendissolved in purified water, preferably gives a solution of pH 4.0 to7.5, more preferably pH 4.5 to 7.0. It is preferable that the thusprepared composition in lyophilized form is sealed and stored withshading. The lyophilized composition can be loaded in each vial in thesolution form before lyophilizing or in lyophilized powder form afterlyophilizing.

Since the cyclic polypeptide compound is not satisfactorily stable tohumidity, it is necessary that the lyophilized composition of thepresent invention contains 3.4% by weight or less of water, preferably3.0%, more preferably 2.0%.

Usually the stabilized pharmaceutical composition in lyophilized form isdissolved in isotonic sodium chloride solution as required and used asan injection solution. The pharmaceutical composition of the presentinvention may be used as an injection preparation which requires somecompounding before use.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is now described in further detail by way ofexamples and test examples, which should not be construed to limit thescope of the invention. In the examples, the compounds of formula (II)to (VI) are referred to as Compounds (II) to (VI), respectively.

EXAMPLE 1

-   Compound (II) 25 g-   Lactose 200 g-   anhydrous Citric acid in a suitable amount-   Sodium hydroxide in a suitable amount

Lactose was dissolved in purified water (2000 ml) under heating below50° C. After cooling below 20° C., the lactose solution was added withCompound (II) avoiding bubbling under gently stirring. After adding 2%aqueous citric acid solution (9.5 ml), the solution was added with 0.4%aqueous sodium hydroxide solution (about 24 ml) to adjust pH 5.5followed by diluting with purified water to make a given volume (2500ml). The resulting solution was dispensed into 1,000 vials of 10 mLvolume, 2.5 ml per vial. The solution in the respective vials waslyophilized by using the lyophilizer (RL-603BS manufactured by KyowaShinku Co., Ltd) by the conventional method to obtain lyophilizedcompositions each containing 25 mg of Compound (II).

EXAMPLE 2

Lyophilized compositions each containing 50 mg of Compound (II) wereobtained in the same manner as in Example 1 except that the amount ofCompound (II) used was 50 g.

EXAMPLE 3

Lyophilized compositions each containing 25 mg of Compound (II) areobtained in the same manner as in Example 1 except that 150 g of maltoseis used instead of lactose.

EXAMPLE 4

Lyophilized compositions each containing 50 mg of Compound (II) areobtained in the same manner as in Example 1 except that the amount ofCompound (II) used is 50 g instead of 25 g and 250 g of sucrose is usedinstead of lactose.

EXAMPLE 5

Lyophilized compositions each containing 25 mg of Compound (II) areobtained in the same manner as in Example 1 except that 25 g of sodiumchloride is used instead of lactose.

EXAMPLE 6

Lyophilized compositions each containing 10 mg of Compound (II) areobtained in the same manner as in Example 1 except that the amount ofCompound (II) used is 10 g instead of 25 g and 100 g of dextran is usedinstead of lactose.

EXAMPLE 7

Lyophilized compositions each containing 25 mg of Compound (III) areobtained in the same manner as in Example 1 except that 25 g of Compound(III) is used instead of Compound (II) and 200 g of maltose is usedinstead of lactose.

EXAMPLE 8

Lyophilized compositions each containing 10 mg of Compound (IV) areobtained in the same manner as in Example 1 except that 10 g of Compound(IV) is used instead of Compound (II) and the amount of lactose used is100 g instead of 200 g.

EXAMPLE 9

Lyophilized compositions each containing 50 mg of Compound (V) areobtained in the same manner as in Example 1 except that 50 g of Compound(V) is used instead of Compound (II) and 50 g of sodium chloride is usedinstead of lactose.

EXAMPLE 10

Lyophilized compositions each containing 10 mg of Compound (VI) areobtained in the same manner as in Example 1 except that 10 g of Compound(VI) is used instead of Compound (II) and 100 g of dextran is usedinstead of lactose.

Test Example 1

Effect of Stabilizer in Stabilizing Lyophilized Compositions of Compound(II)

10 mg of Compound (II) and, as a stabilizer, 100 mg of lactose or 9 mgof sodium chloride were dissolved completely in 1 ml of water. Theresulting solutions were lyophilized and maintained at 70° C. in glassvials. Nine days after, the resulting compositions were tested on theirappearance, the residual amount of Compound (II), and others. As acontrol, used was a solution of Compound (II) without any stabilizers.The results are shown in Table 1.

TABLE 1 Stabilizers Test Items 0 hours After 9 days Control: AppearanceWhite mass Slightly yellow mass nil pH*  7.1  2.7 Residual amount (%)100.0  8.0 Water content (%)  1.3 — Lactose Appearance White mass Whitemass (100 mg) pH*  6.4  6.1 Residual amount (%) 100.0 99.5 Water content(%)  1.0 — Sodium Appearance White mass White mass chloride pH*  6.7 6.3 (9 mg) Residual amount (%) 100.0 75.9 Water content (%)  0.7 — *pHof reconstituted solutions of compositions in 1 ml of water

Test Example 2

The similar tests were conducted in the same manner as in Test Example 1except that 100 mg of maltose, 50 mg of sucrose or 50 mg of glucose wasused as a stabilizer. The results are shown in Table 2.

(to be continued on the next page)

TABLE 2 Stabilizers Test Items 0 hours After 9 days Control: nilAppearance White mass White mass pH*  6.8    5.4 Residual amount (%)100.0 <75.0 Water content (%)  3.3 — Maltose Appearance White mass Whitemass (100 mg) pH*  7.3    6.7 Residual amount (%) 100.0   98.6 Watercontent (%)  0.9 — Sucrose Appearance White mass White melt (50 mg) pH* 6.9    7.0 Residual amount (%) 100.0   82.4 Water content (%)  1.1 —Glucose Appearance White melt Brown melt (50 mg) pH*  6.9    3.6Residual amount (%) 100    1.1 Water content (%)  4.3 — *pH ofreconstituted solutions of compositions in 1 ml of water

As is obvious from Tables 1 and 2, the lyophilized composition ofCompound (II) and lactose, sodium chloride, maltose or sucrose wassignificantly stable as compared with the one not containing anystabilizers or containing other stabilizers.

Test Example 3

Dependence of the Stability of Lyophilized Compositions of Compound (II)upon the Amount of Lactose Added

Tests were carried out in the same manner as in Test example 1 exceptthat 20 mg, 50 mg, 100 mg or 200 mg of lactose were added as astabilizer. Table 3 shows the results of tests by observation of theappearance of compositions, the residual amount of Compound (II), theappearance of reconstituted solutions of compositions in 1 ml of water,and the like. Incidentally, it took 15 seconds to reconstitute thecompositions in 1 ml of water.

TABLE 3 Amount After 3 of months at lactose After 40° C. and a added 9days 75% (mg) Test Items 0 hours at 70° C. humidity 20 Appearance WhiteSlightly White mass mass yellow mass Color* Colorless White ColorlessClarity* Clear Not clear Clear pH*  6.09  3.03  6.57 Residual amount (%)100.0 88.09 100.0 Total impurities (%)  3.44 12.3  3.99 Water content(%)  1.2 — — 50 Appearance White White White mass mass mass Color*Colorless Colorless Colorless Clarity* Clear Clear Clear pH*  6.57  5.56 6.26 Residual amount (%) 100.0 96.7  99.8 Total impurities (%)  3.32 7.37  4.21 Water content (%)  0.5 — — 100 Appearance White White Whitemass mass mass Color* Colorless Colorless Colorless Clarity* Clear ClearClear pH*  6.58  6.08  5.80 Residual amount (%) 100.0 96.7  99.6 Totalimpurities (%)  3.43  7.08  3.96 Water content (%)  0.3 — — 200Appearance White White White mass mass mass Color* Colorless ColorlessColorless Clarity* Clear Clear Clear pH*  6.78  5.70  5.36 Residualamount (%) 100.0 96.1  99.6 Total impurities (%)  3.40  7.30  4.35 Watercontent (%)  0.3 — — *Color, clarity and pH of reconstituted solutionsof compositions in 1 ml of water

As is obvious from Table 3, the lyophilized compositions of 10 mg ofCompound (II) and various amount of lactose had no problem in theirstability.

Test Example 4

Stability of Lyophilized Compositions of 200 mg of Lactose and Compound(II) in Vial

Tests were carried out in the same manner as in Test Example 1 exceptthat 12.5 mg, 25 mg, 50 mg, 75 mg or 100 mg of Compound (II) were usedwith 200 mg of lactose. Table 4 shows the results of the tests on theresidual amount of Compound (II) in the resulting compositions and thelike. Regarding all the compositions, their appearance is a white mass,the time for reconstitutional dissolution was 15 seconds, and the colorand the clarity of reconstituted solutions of the compositions werecolorless and transparent.

TABLE 4 Amount After 3 of months Com- at 40° C. pound and (II) After 9After 21 a 75% added days at days at humid- (mg) Test Items 0 hours 70°C. 60° C. ity 12.5 pH* 6.63 6.15 6.31 6.08 Residual amount (%) 100.098.1 97.5 99.6 Total impurities (%) 2.24 3.95 3.75 2.71 Water content(%) 1.3 — — — 25 pH* 6.37 6.07 6.11 6.14 Residual amount (%) 100.0 99.398.2 101.2 Total impurities (%) 2.25 4.03 3.49 2.68 Water content (%)1.1 — — — 50 pH* 6.26 5.99 6.00 6.00 Residual amount (%) 100.0 97.9 97.3100.5 Total impurities (%) 2.25 3.95 3.68 2.74 Water content (%) 1.2 — —— 75 pH* 6.13 5.95 5.96 6.04 Residual amount (%) 100.0 98.1 97.7 99.0Total impurities (%) 2.28 4.14 3.83 2.76 Water content (%) 0.9 — — — 100pH* 6.03 5.92 5.88 5.85 Residual amount (%) 100.0 97.8 96.7 99.5 Totalimpurities (%) 2.46 4.15 3.92 2.79 Water content (%) 1.3 — — — *pH ofreconstituted solutions of compositions in 5 ml of purified water

As is obvious from Table 4, all the lyophilized compositions werestable.

Test Example 5

Stability Test The pharmaceutical compositions obtained in Examples 1and 2 were stored at room temperature. After 18 months, the residualratio of Compound (II) was 98% in all the compositions.

Test Example 6

Dependence of the Stability of Lyophilized Compositions of Compound (II)upon the pH Value of the Solution of the Composition before Lyophilizing

10 mg of Compound (II) and, as a stabilizer, 100 mg of lactose weredissolved completely in 1 ml of citrate-NaOH buffer having different pHvalue between pH 4.0 to 7.0. The resulting solutions having different pHvalues were lyophilized and maintained at 70° C. in glass vials. Ninedays after, the resulting compositions were tested on their pH and theresidual amount of Compound (II). The results are shown in Table 5.

TABLE 5 pH of the solution of the composition before lyophilizing 4.04.5 5.0 5.5 6.0 6.5 7.0 0 pH* 3.9 4.4 4.8 5.4 5.8 6.4 6.8 hours Water0.2 0.2 0.3 0.3 0.3 0.4 0.3 content (%) Residual 100 amount (%) 9 pH*4.0 4.5 4.9 5.4 5.8 6.4 6.8 days Residual 94.4 95.9 97.4 98.5 97.7 96.995.8 amount (%) *pH of reconstituted solutions of compositions in 5 mlof purified water

As is obvious from the table 5, the pharmaceutical composition of thepresent invention is stable after lyophilizing the solution containingCompound (II) at pH 4.0 to 7.0 at least, preferably at pH 4.5 to 7.0.

Test Example 7

Dependence of the Stability of Lyophilized Compositions of Compound (II)upon the Water Content of the Composition

10 mg of Compound (II) and, as a stabilizer, 50 mg of lactose weredissolved completely in 1 ml of water. The resulting solutions werelyophilized and maintained at 70° C. in glass vials. Nine days after,the resulting compositions were tested on their pH, their water contentand the residual amount of Compound (II). The results are shown in Table6.

TABLE 6 Water content at 0 hours (%) 0.9 1.4 2.6 3.4 5.1 0 hours pH* 7.1After 9 days pH* 7.5 7.1 6.8 6.8 3.5 Water content 2.5 2.9 3.6 4.3 5.4(%) Residual 97.6 98.1 97.1 92.7 18.3 amount (%) *pH of reconstitutedsolutions of compositions in 1 ml of water

As is obvious from Table 6, the pharmaceutical composition of thepresent invention is stable containing about 3.5%, more particularly3.4% by weight or less of water.

According to the present invention, provided is a composition inlyophilized form in which the cyclic polypeptide compound (I) or itspharmaceutically acceptable salt are stabilized by a stabilizer such aspolysaccharide, disaccharide and sodium chloride.

The mechanism of the stabilization of the cyclic polypeptide compound(I) or its pharmaceutically acceptable salt by the stabilizer such aspolysaccharide, disaccharide and sodium chloride is still to be unknown,but it may be that the stabilizer adsorbs water in lyophilized cakes andthat the stabilizer serves to disperse the compound or itspharmaceutically acceptable salt uniformly in the composition.

The cyclic polypeptide compound (I) has an antifungal activity,particularly against the following fungi.

Acremonium;

Absidia (e.g., Absidia corymbifera, etc);

Aspergillus (e.g., Aspergillus clavatus, Aspergillus flavus, Aspergillusfumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus,Aspergillus versicolor, etc);

Blastomyces (e.g., Blastomyces dermatitidis, etc);

Candida (e.g., Candida albicans, Candida glabrata, Candidaguilliermondii, Candida kefyr, Candida krusei, Candida parapsilosis,Candida stellatoides, Candida tropicalis, Candida utilis, etc.);

Cladosporium (e.g., Cladosporium trichoides, etc);

Coccidioides (e.g., Coccidioides immitis, etc);

Cryptococcus (e.g., Cryptococcus neoformans, etc);

Cunninghamella (e.g., Cunninghamella elegans, etc);

Dermatophyte;

Exophiala (e.g., Exophiala dermatitidis, Exophiala spinifera, etc);

Epidermophyton (e.g., Epidermophyton floccosum, etc);

Fonsecaea (e.g., Fonsecaea pedrosoi, etc);

Fusarium (e.g., Fusarium solani, etc);

Geotrichum (e.g., Geotrichum candiddum, etc);

Histoplasma (e.g., Histoplasma capsulatum var. capsulatum, etc);

Malassezia (e.g., Malassezia furfur, ec);

Microsporum (e.g., Microsporum canis, Microsporum gypseum, etc);

Mucor;

Paracoccidioides (e.g., Paracoccidioides brasiliensis, etc);

Penicillium (e.g., Penicillium mameffei, etc);

Phialophora;

Pneumocystis (e.g., Pneumocystis carinii, etc);

Pseudallescheria (e.g., Pseudallescheria boydii, etc);

Rhizopus (e.g., Rhizopus microsporus var. rhizopodiformis, Rhizopusoryzae, etc);

Saccharomyces (e.g., Saccharomyces cerevisiae, etc);

Scopulariopsis;

Sporothrix (e.g., Sporothrix schenchii, etc);

Trichophyton (e.g., Trichophyton mentagrophytes, Trichophyton rubrum,etc);

Trichosporon (e.g., Trichosporon asahii, Trichosporon cutaneum, etc).

The above fungi are well known to cause various infection diseases inskin, hair, nail, oral mucosa, gastrointestinal tract, bronchus, lung,endocardium, brain, meninges, urinary organ, vaginal protion, oralcavity, ophthalmus, systemic, kidney, bronchus, heart, external auditorycanal, bone, nasal cavity, paranasal cavity, spleen, liver, hypodermaltissue, lymph duct, gastrointestine, articulation, muscle, tendon,interstitial plasma cell in lung, and so on.

Therefore, the cyclic polypeptide compound (I) of the presentcomposition is useful for preventing and treating various infectiousdiseases, such as dermatophytosis (e.g., trichophytosis, etc),pityriasis versicolor, candidiasis, cryptococcosis, geotrichosis,trichosporosis, aspergillosis, penicilliosis, fusariosis, zygomycosis,sporotrichosis, chromomycosis, coccidioidomycosis, histoplasmosis,blastomycosis, paracoccidioidomycosis, pseudallescheriosis, mycetoma,mycotic keratitis, otomycosis, pneumocystosis, and so on.

A commercial package comprising the cyclic polypeptide compound (I) ofthe present composition and a written matter associated therewith,wherein the written matter states that the pharmaceutical compositioncan or should be used for preventing or treating infections disease.

1. A stabilized pharmaceutical composition in lyophilized form whichcomprises a cyclic polypeptide compound of the general formula (I):

wherein R¹ is a hydrogen atom or an acyl group and R² and R³ are, thesame or different, a hydrogen atom or a hydroxyl group; or itspharmaceutically acceptable salt; and one or more compound(s) selectedfrom the group consisting of a polysaccharide, a disaccharide and sodiumchloride.
 2. The composition according to claim 1 in which R¹ isrepresented by the formula:

and R² and R³ are hydroxy groups.
 3. The composition according to claim1, wherein said compound is a disaccharide.
 4. The composition accordingto claim 1, wherein said compound is maltose or sucrose.
 5. Thecomposition according to claim 1, wherein said compound is lactose. 6.The composition according to claim 1, which contains 0.4 to 50 parts byweight of said compound(s) with respect to one part by weight of thecyclic polypeptide compound or its pharmaceutically acceptable salt. 7.The composition according to claim 1, which contains 0.1 to 400 mg ofthe cyclic polypeptide compound or its pharmaceutically acceptable saltin a single unit dose.
 8. The composition according to claim 1 preparedby: dissolving the cyclic polypeptide compound (I) or itspharmaceutically acceptable salt, the said compound(s), and optionally apH adjustor, in a purified water and lyophilizing the solution.
 9. Thecomposition of claim 1 which when dissolved in purified water, gives asolution of pH 4.0 to 7.5.
 10. The composition of claim 1 which contains3.4% or less by weight of water.
 11. A method for preparing a stabilizedpharmaceutical composition comprising: dissolving a cyclic polypeptidecompound of the general formula (I):

wherein R¹ is a hydrogen atom or an acyl group and R² and R³ are, thesame or different, a hydrogen atom or a hydroxyl group, or itspharmaceutically acceptable salt and one or more compounds selected fromthe group consisting of a polysaccharide, a disaccharide and sodiumchloride in water, and optional a pH adjustor, and lyophilizing thesolution.
 12. An injection preparation prepared by dissolving thecomposition of claim 1 in isotonic sodium chloride solution.
 13. Acommercial package comprising: the pharmaceutical composition of claim 1and written instructions for use thereof.
 14. The composition of claim1, wherein said compound is a polysaccharide.
 15. The composition ofclaim 1, wherein said compound is sodium chloride.
 16. The compositionof claim 1, further comprising a pH adjustor.
 17. The composition ofclaim 16, wherein the pH adjustor is acidic.
 18. The composition ofclaim 16, wherein the pH adjustor is basic.
 19. An aqueous compositioncomprising the composition of claim 1 and water.
 20. The aqueouscomposition of claim 19, wherein the water is present in an isotonicsodium chloride solution.
 21. The aqueous composition of claim 19,wherein the water consists essentially of purified water.
 22. A methodfor treating a fungal disease comprising administering an effectiveamount of the composition of claim 1 to a subject in need thereof. 23.The method of claim 22, wherein said disease is selected from the groupconsisting of dermatophytosis, pityriasis versicolor, candidiasis,cryptococcosis, geotrichosis, trichosporosis, aspergillosis,penicilliosis, fusariosis, zygomycosis, sporortrichosis, chromomycosis,coccidioidomycosis, histoplasmosis, blastomycosis,paracoccidioidomycosis, pseudallescheriosis, mycetoma, mycotickeratitis, otomycosis and pneumocystosis.